Process of manufacturing diastases and toxins by oxidizing ferments.



A. BOIDIN & J. EFFRONT. PROCESS OF MANUFACTURING DIASTASES A ND TOXINS BY OXIDIZING FERMENTS;

APPLICATION FILE D JULY 5, I914 Patented May 22; 1917.

'nwm To all whom it may concern:

a no.

"AUGUSTE BOID IN," OF SEGLIN', FRANCE, AND JEAN EFFRON'I', OF BRUSSELS, BELGIUM.

-PROGESS OF MANUFACTURING DIAS'IASES AND TOXINS BY OXIDIZIN G- FERMENTS.

,2 ,5 5, Specification o Letters Patent. t t Maw 22, 19m,

Application filed July. 6, 1914. Serial No. 849,151.

Be it known that we, AUGUsT BOIDIN, a citizen ;of the Republic of France, residing at Seclin, Nord, France, and JEAN EFFRONT, a subject of the King of Belgium, residing at "73 Avenue Solbosch', Brussels, Belgium,

, have invented new and useful Improvements in Processes of Manufacturing Diastases and Toxins by Oxidizing Ferments, of

which the following is a specification.

In studying the liquefaction of starchy andnitrogenous substances by means" of bacteria in an alkaline medium, we have met with a series of difficulties in endeavoring to rice, maize,potato, or'barley, on the one hand, and on soy cake of 10 per cent.

strengthen the other hand, it is found that the wort of soy yields from 8 to 10 times the amount of diastase obtained from the media previously named.

Now, we have established the fact that the diastasic power is proportional to the amount of nitrogen in an assimilable state in the raw, material, that all raw materials pass'from laboratory to industrial working. which only contain about one part of nitro- The bacteria which, in the small vessels gen to 35 to 45 parts of carbohydrates at the used in the laboratory, gave fairly active somost yield solutions of diastase of low aclutions, refused to secrete diastases when tivity,' in which the secretion of enzyms transferred to culture apparatus of larger is retarded. Conversely, raw materials dimensions. which contain one part of nitrogen to every The study of these difiiculties has led to' 10 or 15 parts of carbohydrates yield solu the recognition of their causes, and at the tions which are very rich in diastases; same timeto the discovery of the rules to be among these may be mentioned, soy, and esobserved in order to bring about an abunpecially soy cake from which the fat has dant secretions of diastases, and a diastasic been extracted; vegetable or animal albumiactivity such as it has not been possible to noid materials, caseins, the fibrin of maize, attain heretofore. v, etc., may also be used.

The study of the influence of metals on A certain number of aerobic bacteria live i the secretion of bacterial enzyms has shown very badly in the depth, even if the wort that the multiplication of oxidizing bacteria be constantly traversed by a strong current growing in media rich in' nitrogenous substances 1s the more hampered by the pres ence of. certain metals the more considerable the amount of, oxygen supplied to the bac 3.

of air, and yield under these conditions acid solutions very poor in diastases. Cultivated in immediate contact with air, these same bacteria yield alkaline solutions rich in diateria and that, other things beingequalg the stases when the nutrient material is sufli production of diastase varies with the nature of the metal in the presence of--which the culturesare carried on. I

Thus, a series of 96-hour cultures, on a ort of soy at 10 per cent.', gives the following liquefying powers 100 in glass 83 in tinned iron 25 in. galvanized iron 25 in tinned copper 14 in iron 12 in copper 3 in presence of copper in thin sheets J '100 in enameled metals 100 in aluminium. Now, wehave recognized ,.that. to obtain bacteria, diastases amylolytic or proteolytic,

'"ciently rich in nitrogen.

paratus described-below for carrying out the process areapplicable to all bacteria which possess the two properties ust described.

In order to get a good diastasic power it is necessary so to arrange the conditions that the growth remains on the surface of the liquid aiid forms a. film there. For those species which form afilm'with difliculty it is advantageous to inoculate concentrated wort, or such as contain solid bodies, and on the surface.

The direct contact of the bacteria with of the liquid isnot competent to determine different diastases withthe most 'diverse the-production of diastase when the growth remains submerged.

At the commencement of v mcubat1on, 1t

is advantageous to give much air, but when the film begins to thicken the aeration must be reduced as nearly to the minimum as possible, in order that the yield of diastase acid may not fall.

The surface aeration must be so elfected that the carbonic acid may be able to escape steadily, and the apparatus must be so arranged as that the aeration may be regulated to a point at which analysis shows that the escaping gas does not contain too much. carbonic acid. The object of the aeration from above downward is that it may be able to be kept under control at the end of culture.

Cultures kept absolutely at rest form diastases slowly, their production is accelerated by gentle shaking or mechanical motion, which must be such that submersion is avoided. The diffusion of the active substances and the supply of fresh nutrient matter are thus facilitated.

It will be understood that the form of the apparatus employed for carrying out the process constituting this invention may be infinitely varied.

When it is a question of important instal lations, preference will be given to the following arrangements, which allow lar e quant1ties of hquefying diastases to be 0 tained without. appreciable cost for labor, the filling, the emptying, and the cleaning being then mechamcally efiected by centrifugal force.

We proceed to' describe, by way of example, the arrangement which appears the most convenient, and the most certain to secure asepsls.

In the appended this arrangement in elevation, partially broken away, and Fig. 2 in plan.

In 1 there is a sterilizing autoclave, resistant to pressure, furnished with a steam pipe 2, which may also serve for theemptying, a tubulure 3 intended to receive the inoculating tap and another tubulure 4 which carries the safety valve 5, the pressure gage 6 and a clearing cock 7. The autoclave is provided with a hand agitator.

At 8 there is a cotton wool filter to sterilize the air under pressure which .enters at 9; the cocks 10 and 11 serve for the introduction of steam, and 11 carries a small clearing cook 12. At 13 is a tap which serves successively for steam and for pure air; it is in communication with the air moistener 14 and the culture chamber 15 by the aeration pipe'16; 17 and 18 are two cocks necessary for the sterilization of the wort in 1.

The culture chamber 15 is composed of an aluminium reservoir, with a diameter of 1.0 to 1.50 meters, which may be sterilized under a pressure of one kilo. Itis fur nished at its upper part with a tubulure for the receipt of the charging pipe 2, with another tubulure connected to the air drawing Figure 1 shows 7 supply pipe 16, with others forthe safety valve and the pressure gage. It is also provided below with an aluminium pipe 19 having a cock 20 and a tube 21 dipping into a water vessel 22.

The upper lid is provided with a stufling box and carries a gearing which turns the vertical shaft 23, mounted in a step hearing 24. This gearing may consist, for example, of a spindle 33 turned by a handle 34 and connected to the shaft 23 by bevel pinions 35, 36.

The shaft 23 supports a series of plates 25 of a thickness of 1 or 2 mm. kept parallel by central washers 26 and by others threaded on rods 27. These washers are pressed at 28 between two strong plates of considerable thickness, in such a way as to maintain the perfect horizontality of the plates. Finally each plate has a raised border, 10 to 20 mm. high, according to the thickness of the layer of liquid which it is thought desirable to adopt and of an overflow 29.

To charge the apparatus, we proceed as follows:

Through the manhole of the cooker 1 are introduced the water and say, the soy cake. These are heated to boiling by passing in steam through 2, the orifices 3 and 4 being open and the cook 17 also slightly open to insure the sterilization of the upper part of the tube 2. At the same time steam is injected through 13, 14: and 16 in such a way as to bathe with steam the apparatus 15,

the cock 20 being open at first and the vessel 22 empty.

The contents of the cooker are heated for about 30 minutes under a pressure of 1 kilo, the agitator being turned at the same time in such a way as to insure the absolute sterility of the wort and its homogeneity.

During this time, the container 15 with its plates has been sterilized, together with the cock 2O (nearly closed) by steam under pressure, and the plates are so turned as to get rid through 20 of the condensed water with which they have become loaded. The steam is then replaced by air under the precautions known by all experts who have had charge of apparatus for the pure culture of yeast, or of those known by the name of amylo apparatus.

' The sterilizer is put under pure air presteria. The air is thereafter reduced to a ,the development of the To insure the equitable distribution of the Wort on the plates, it is advisable to impart to them a slight' "to-and -fro motion.

The emptying of the sterilizer being effected, the room in which the apparatus is placed is kept at a temperature such that the thermometers 31 and 32 remain within the limits of the optimum temperatures for particular organism At the beginning aeration is carried on pretty strongly, and this is continued until the bacteria form a continuous layer over the surface of each plate. A to andfro motion is imparted several times a day to dissociate the chains of bacunder treatment.

strict minimum, and the analysis of the gases issuing at 22will indicate, by the oxygen content, whether enough isbeing furnished to the bacteria.

When the culture has acquired its maxi mum dlastasic power, which can be determined by taking a sample at=19, after having turned the plates slightly the apparatus is emptied by centrifugal force by opening 20. The apparatus is-then washed with water, this water, like the solution of diastase, leaving the plates by centrifugal force.

. A variant of the process consists in cultivating the bacteria continuously, that is to say, in emptying the apparatus more or less completely after the first culture and adding cooled wort in such a way that it inoculates itself with the bacteria left upon the plates.

Thanks to the arrangements above described and the'keeping of the rules laid down, there are obtained in two or three days diastase of great potency, 200 to 300 liters suflicing to liquefy 100 tons of grain.

The advantages of the apparatus described are the simplicity of construction, great surface with limited volume, simplicity of manipulation and great economy of labor.

It is evident from What precedes that the arrangement of the apparatus may be infi nitely varied, that the vertical cylinder 15 may, for instance, be replaced by a horizontal one, that the mode of fixing the plates may vary according as one uses those of enamel, aluminium,- porcelain, glass, etc., that instead of rotating plates, and its envelop might be made to turn, or it might be made to oscillate to insure the emptying of the plates; moreover, one might construct the apparatus of a pile of independent plates, to make the culture on solid media such as soy cake with 1 or?! parts of water. The cylinder 15 might also be filled with pumice-stone, coke, shavings, etc, but experience has shown that culture on these or other inertbodies and in extra thin layers only gives a feeble diastatic power.

Cultures of diastase produced by the. aid

the apparatus of raw materials rich in albumin can easily be preserved in air by the addition ofvapor antiseptics, such as 10 to 15 percent. of sea salt, 4 to 5 per mille of the 40 per cent. forinol of commerce, sulfate of magnesia or ammonia, etc.

The diastasic power of these solutions so preserved in corked barrels rises slightly with time. Finally, with the same object of preservation, the diastases or toxinsmay be precipitated by a salt such as sulfate of ammonia, or the solutions of diastase evaporated in cacao. By evaporation in oaouo or in the air, an increase of the diastasic power is obtained.

Claims:

1. A process for the manufacture of diastases and toxins by oxidizing ferments which consists in using as a nutritive medium a wort containing at least one part of assimilable nitrogenous materials to fifteen parts of carbohydrates, and cultivating bacteria at the surface of the said wort in thin layers.

2. A process for the manufacture of diastases and toxins by oxidizing ferments consisting in using as a nutritive medium a wort containing at least one part of assimilable nitrogenous materials to fifteen parts of carbohydrates, and in cultivating bacteria at the surface of the said wort in layers, the thickness of which is not more than eight millimeters.

s 8. A process for the manufacture of diastases and toxins by oxidizing ferments consisting in cultivating bacteria at the surface of a wort containing assimilable nitrogenous materials, and subjecting said wort to a slight agitation, said agitation being sulficient to break the associations of bacteria arranged. in chain-like lines.

4. A process for the manufacture of (imstases and toxins by oxidizing ferments consisting in cultivating bacteria at the surface of a WOI't containing nitrogenous materlals spread in thin'layers on horizontal plates 5. A process for the manufacture of dlastases and toxins by oxidizing ferments consisting in cultivating bacteria at the surface of a wort containing nitrogenous materlals in an atmosphere which isventilated less and less as the bacteria produce less and less carbonic dioxid. V I

6. A process for the manufacture of diastases and toxins by oxidizing ferments consisting in cultivating bacteria at the surface of a wort of soj a deprived of fat.

AUGUSTE Bonnu'. JEAN EFFRONT.

Witnesses:

-CHAs. ROY NASMITH, ALFRED DE LARVILLE. 

